The Biosynthesis of Lipooligosaccharide from Bacteroides thetaiotaomicron

ABSTRACT Lipopolysaccharide (LPS), a cell-associated glycolipid that makes up the outer leaflet of the outer membrane of Gram-negative bacteria, is a canonical mediator of microbe-host interactions. The most prevalent Gram-negative gut bacterial taxon, Bacteroides, makes up around 50% of the cells in a typical Western gut; these cells harbor ~300 mg of LPS, making it one of the highest-abundance molecules in the intestine. As a starting point for understanding the biological function of Bacteroides LPS, we have identified genes in Bacteroides thetaiotaomicron VPI 5482 involved in the biosynthesis of its lipid A core and glycan, generated mutants that elaborate altered forms of LPS, and used matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry to interrogate the molecular features of these variants. We demonstrate, inter alia, that the glycan does not appear to have a repeating unit, and so this strain produces lipooligosaccharide (LOS) rather than LPS. This result contrasts with Bacteroides vulgatus ATCC 8482, which by SDS-PAGE analysis appears to produce LPS with a repeating unit. Additionally, our identification of the B. thetaiotaomicron LOS oligosaccharide gene cluster allowed us to identify similar clusters in other Bacteroides species. Our work lays the foundation for developing a structure-function relationship for Bacteroides LPS/LOS in the context of host colonization.

or cell wall that are architecturally similar although chemically different among bacterial taxa-lipopolysaccharides (LPSs), lipoteichoic and wall teichoic acids, mycolic acids, and muramyl dipeptides are key examples. Their ubiquity on the cell surface makes them excellent targets for bacterial detection by innate immune receptors, including Toll-like receptors and NOD proteins (2). However, a longstanding question remains: how do innate immune cells "know" whether the bacterial cell that they encounter is a mutualist or a pathogen and "decide" how to respond appropriately? Part of the answer likely involves unique strain-specific chemical signatures within these cellassociated molecules.
Lipopolysaccharide (LPS) is a canonical cell-associated glycolipid. The interaction between LPS and host Toll-like receptor 4 (TLR4) is a paradigm for immunologic sensing of Gram-negative bacteria. LPS is generally composed of a lipid anchor (termed lipid A), a core oligosaccharide region, and a polysaccharide repeating unit called the O antigen. The core oligosaccharide and O antigen are typically biosynthesized from separate gene clusters, while lipid A biosynthetic genes are distributed throughout the genome (3). The chemical structure of LPS varies considerably among species, and these differences in structure are relevant to function. For example, Yersinia pestis deacylates its lipid A when infecting humans, thus avoiding detection by TLR4 (4). Helicobacter pylori elaborates its O antigen with Lewis antigens to mimic host glycans (5,6). More drastic changes in overall structure have also been observed. Species of Neisseria produce an LPS variant, known as lipooligosaccharide (LOS), which has a more elaborate core oligosaccharide in place of the conventional O antigen (7,8). Notably, almost everything known about the biosynthesis, structure, and function of LPS comes from studies of "conventional" pathogens. Remarkably little is known about LPS from commensal organisms and its importance to host innate immunity.
Among the glycolipids found in the gut microbiome, Bacteroides LPS is of particular interest. Bacteroides and, in~10% of humans, its relative Prevotella are the only high-abundance Gram-negative bacterial genera in the gut. Bacteroides as a genus makes up~50% of the typical Western gut community (9). Notably, the species distribution within that 50% is highly variable between individuals (10). Different Bacteroides species have been reported to produce LPS molecules with distinct architectures based on their banding pattern on an SDS-PAGE gel, suggesting that each Bacteroides species has the potential to influence innate immunity in its own way (11,12). Bacteroides LPS is already known to have a different lipid A structure than "pathogenic" LPS: Bacteroides thetaiotaomicron, Bacteroides fragilis, and Bacteroides dorei produce penta-acylated, monophosphorylated lipid A, in contrast to the hexa-acylated, diphosphorylated lipid A from Escherichia coli (13)(14)(15)(16). With a recent exception reporting a B. thetaiotaomicron lipid A phosphatase, very little is known about the biosynthetic genes involved in Bacteroides LPS biogenesis (17).
It takes as little as 50 ng of E. coli LPS injected intravenously into a mouse to cause septic shock (18). In contrast, given our laboratory purification yield of approximately 10 mg B. thetaiotaomicron LPS per 1 liter of confluent culture and assuming~7 ϫ 10 11 bacteria per liter in vitro and 20 trillion Bacteroides cells per individual, we estimate that a typical Western human gut contains~300 mg of Bacteroides LPS, likely making it one of the highest-abundance bacterially derived molecules present (19). We set out to better define the biosynthesis and structure of Bacteroides LPS as a starting point for understanding and manipulating the interaction between Bacteroides and the mammalian immune system.

RESULTS AND DISCUSSION
Characterization of the Bacteroides lipid A core. In order to identify candidate biosynthetic genes for Bacteroides lipid A, we performed BLAST searches against Bacteroides genomes using, as queries, the E. coli MG1655 lipid A biosynthesis genes (20). E. coli normally produces a lipid A molecule that has six acyl chains and two phosphate groups, as shown in Fig. 1A labeled "Kdo 2 -lipid A." As expected, orthologs of each Raetz pathway enzyme were identified, except that the Bacteroides species had pathway, where starting material UDP-GlcNAc is acylated, glycosylated, and phosphorylated by a series of nine biosynthetic enzymes to produce 3-deoxy-D-manno-octulosonic acid 2 (Kdo 2 )-lipid A. E. coli produces lipid A that is phosphorylated at both the 1 and 4= positions on the diglucosamine backbone, but the lipid A 1-and 4=-phosphatases LpxE and LpxF have been identified in other bacteria and are thought to act after biosynthesis of Kdo 2 -lipid A is complete. (B) Locus tags of (Continued on next page) Bacteroides Lipooligosaccharide Biosynthesis ® only one ortholog of the acyltransferases LpxL and LpxM; we refer to this ortholog as LpxL for simplicity. LpxL and LpxM are responsible for adding the fifth and sixth acyl chains to E. coli lipid A, so the presence of only one of these acyltransferases in Bacteroides genomes is consistent with published reports that B. thetaiotaomicron, B. fragilis, and Bacteroides dorei lipid A is penta-acylated rather than hexa-acylated (13)(14)(15). Bacteroides vulgatus was the only surveyed species to have a second LpxL/LpxM homolog, BVU_1014. Previous work indicates that this gene is part of an aryl polyene gene cluster, indicating that it likely transfers an acyl chain to a non-LPS substrate (21). We next used BLAST to predict lipid A phosphorylation by sequence homology to the lipid A 1-and 4=-phosphatases discovered in Porphyromonas gingivalis (22,23) (Fig. 1). While this search resulted in only one candidate for some species like B. thetaiotaomicron, for others there were multiple candidates, and experimental validation will be necessary to conclude which, if any, perform the predicted function.
In order to determine the lipid A profile of each species, we isolated lipid A from five common Bacteroides species using the TRI reagent method and characterized their lipid A profile by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) (24). Consistent with previous reports, the structures of B. thetaiotaomicron VPI 5482 and B. fragilis NCTC 9343 lipid A are penta-acylated and monophosphorylated, with their MALDI spectra showing a cluster of peaks around 1,688 m/z (13,14). Moreover, Bacteroides uniformis ATCC 8492, B. vulgatus ATCC 8482, and Bacteroides ovatus ATCC 8483 produce lipid A with virtually identical mass spectra (Fig. 2). Because the bacteria were grown in rich medium under normal anaerobic growth conditions, we cannot be certain that the structure of their lipid A is the same under conditions of host colonization, nor do we know whether it can change in response to stresses encountered in the host.
Characterization of late biosynthetic genes in B. thetaiotaomicron lipid A biosynthesis: acylation and dephosphorylation. Because lipid A is typically an essential component of the outer membrane of Gram-negative bacteria, deletion of genes in the lipid A biosynthetic pathway is frequently lethal to bacteria (25)(26)(27)(28)(29)(30). Interestingly, the later biosynthetic genes such as those for the acyltransferases (lpxL and lpxM) and homologs of the Raetz pathway genes in E. coli MG1655 and lpxE and lpxF in Porphyromonas gingivalis W83 from a selection of Bacteroides species. Bacteroides has homologs for every gene in the pathway except that it has only one secondary acyltransferase, suggesting that its lipid A is predominantly penta-acylated. The species vary more in their putative homologs of the P. gingivalis phosphatases. phosphatases (lpxE and lpxF) can often be deleted (31). Working in B. thetaiotaomicron VPI 5482 ⌬tdk, our background strain for genetic knockouts lacking the thymidine kinase gene tdk, we made scarless single deletions of the putative lpxL and lpxF orthologs that we had identified by BLAST search (BT2152 and BT1854, respectively) and isolated lipid A from the resulting mutants. MALDI-TOF analysis showed a loss of 224 Daltons in the ΔlpxL mutant, consistent with the loss of a 15-carbon acyl chain, and a gain of 80 Daltons in the ΔlpxF mutant, indicating the addition of a phosphate group (Fig. 3). The assignment of BT1854 as the B. thetaiotaomicron lipid A 4=-phosphatase represents independent confirmation of a result first reported by Goodman and coworkers (17).
Lipid A acylation and phosphorylation are important both for bacterial membrane physiology and for the interaction between LPS and host TLR4. An ΔlpxL ΔlpxM double mutant of E. coli MG1655-which lacks the fifth and sixth acyl chains on lipid A, yielding a tetra-acylated, diphosphorylated molecule referred to as lipid IV A -cannot grow above 32°C (Fig. 1A) (32). The crystal structure of the TLR4/MD-2 complex with E. coli lipid A suggests that the number of acyl chains and the number and position of phosphate groups on the molecule may affect binding affinity to the receptor and possibly receptor dimerization (33). E. coli lipid IV A is capable of inhibiting TLR4 activation by wild-type E. coli lipid A (34). This tetra-acylated diphosphorylated scaffold has been used in the design of eritoran, a lipid A mimic developed as a TLR4 antagonist as a potential treatment for sepsis (35). Additionally, lipid A mutants in E. coli differentially stimulate NF-B production in a THP-1 reporter cell line (36). We hypothesize that identifying lipid A biosynthesis genes in Bacteroides will allow us to make mutants that may have different immunostimulatory abilities and could be used to control innate immune responses in a host. Toward this goal, we have generated a B. thetaiotaomicron ΔlpxL ΔlpxF double mutant that elaborates tetraacylated, diphosphorylated lipid A, which we anticipate will be a TLR4 antagonist (see  Bacteroides Lipooligosaccharide Biosynthesis ® O55:B5 and E. coli MG1655 LPS to confirm the previous observation that B. vulgatus produces LPS exhibiting a "laddered" pattern on a gel like E. coli O55:B5 does, whereas B. thetaiotaomicron LPS does not (11,12). The laddering pattern is of note because it indicates the presence of an O antigen; the number of repeating units added to the core oligosaccharide is variable, so the result is a population of LPS molecules of different sizes. As shown in Fig. 4, B. vulgatus LPS appears to have an O antigen based on the laddered pattern of its LPS, but B. thetaiotaomicron instead appears to synthesize a small number of structures that we propose are more likely to be lipooligosaccharides (LOSs) due to their apparent lack of an O antigen. Here, we will refer to the B. thetaiotaomicron outer membrane glycolipid as a LOS rather than LPS.
Recent work on the human-associated bacteria H. pylori and Hafnia alvei indicates that the glycan portion of LPS from these strains can interact with C-type lectin receptors DC-SIGN and dectin-2, respectively, to influence a dendritic cell's cytokine output (37,38). Engagement of this class of receptors by a human-associated bacterium lacking an O antigen has not been investigated, however, and so we sought to identify the biosynthetic route for the oligosaccharide component of B. thetaiotaomicron LOS to explore its apparent lack of an O antigen and provide tools for manipulating the glycan structure.
Identification of the gene cluster for LOS oligosaccharide biosynthesis in B. thetaiotaomicron. Previous work analyzing biosynthetic gene clusters from the NIH Human Microbiome Project data indicated that the phylum Bacteroidetes contains the largest number of predicted saccharide-producing gene clusters (39). B. thetaiotaomicron alone is known to harbor eight gene clusters responsible for making different capsular polysaccharides (CPSs) (40,41). We first wanted to determine whether any of these CPS gene clusters influenced the assembly of LOS. We isolated LOS from a B. thetaiotaomicron mutant constructed by Martens and coworkers in which all eight of its CPS clusters have been deleted (labeled the ΔCPS strain), as well as eight additional strains that each possess only one CPS cluster (CPS1-only, CPS2-only, and so on) (42). By SDS-PAGE analysis, we determined that neither deletion nor expression of CPS clusters affects the banding pattern of B. thetaiotaomicron LOS, indicating that these gene clusters do not encode the biosynthetic machinery for synthesis of B. thetaiotaomicron LOS (Fig. S2).
An independent lead came from a recently published report: by screening a B. thetaiotaomicron transposon library using an antibody that binds the bacterial cell surface of B. thetaiotaomicron, Peterson et al. identified a gene cluster that they predicted might be responsible for the biosynthesis of the B. thetaiotaomicron LPS O antigen, due to a lack of antibody binding when the cluster was disrupted and the annotated functions of several of the genes within the cluster (43). Surprisingly, nine out of the 13 transposon mutants that did not bind the antibody had insertions in genes in the same gene cluster, BT3362 to BT3380 (Fig. 5A). No transposon insertions were obtained in the first three genes of the cluster, indicating that these genes might be essential or their deletion might lead to the accumulation of a toxic intermediate. Intrigued, we obtained a subset of these transposon mutants and analyzed LOS isolated Bacteroides Lipooligosaccharide Biosynthesis from each mutant by SDS-PAGE (Fig. 5B). Each mutant produced LOS with a banding pattern that appeared different from that of the wild type, except for the mutant with an insertion in the final gene in the cluster, BT3380. These data suggest that BT3362 to BT3380 encode the biosynthesis of the B. thetaiotaomicron LOS oligosaccharide. Additionally, they support our hypothesis that bands observed on the SDS-PAGE gel are glycans that do not have a single polymerized repeating unit but rather are variants of a heterogeneous oligosaccharide.
Furthermore, the predicted function of the genes within the BT3362 to BT3380 cluster also supports this conclusion. This cluster bears some resemblance to the waa core oligosaccharide gene clusters characterized in E. coli, with the first gene, BT3362, sharing homology with the genes for heptosyltransferases WaaC and WaaQ (44). Overall, the cluster possesses 13 predicted glycosyltransferases (BT3362-BT3363, BT3365 to BT3372, BT3377, and BT3379-BT3380), a putative LPS kinase (BT3363), four tailoring enzymes (BT3373 to BT3376), and a GtrA-like protein (BT3378). It is unclear what specific structural modifications BT3373 to BT3376 might make based solely on sequence homology. Proteins in the GtrA-like family are typically integral membrane proteins that are thought to play a role in the transport of cell surface polysaccharides (45,46).

Intact LPS MALDI-TOF MS as a diagnostic tool for LOS mutants.
While the SDS-PAGE analysis of LOS from the transposon mutants implicates BT3362 to BT3380 in B. thetaiotaomicron LOS oligosaccharide biosynthesis, we wanted to increase the resolution of our analysis using mass spectrometry (MS). The LPS/LOS bands on an SDS-PAGE gel are approximations of the sizes of molecules in a sample, and using mass spectrometry would allow us to gain a clearer picture of the molecules made by the transposon mutants. Although it is easier to analyze LPS/LOS by MALDI-TOF after removing the O-and/or N-linked acyl chains via hydrazine or hydrogen fluoride treatment, we chose to analyze intact LOS molecules that were not subjected to chemical degradation or derivatization. We reasoned that LOS from B. thetaiotaomicron may contain important functional groups in the oligosaccharide chain that could be removed by these treatments, further complicating our efforts to elucidate detailed structural information about B. thetaiotaomicron LOS.
We adapted a previously published strategy for analyzing intact LPS/LOS by MALDI-TOF (47,48). LPS/LOS analysis is typically challenging due to difficulties in inducing the glycolipid to ionize because of its size and polarity. We chose three transposon mutants that appeared by SDS-PAGE analysis to be truncated to various degrees (B. thetaiotaomicron tn3365, B. thetaiotaomicron tn3368, and B. thetaiotaomicron tn3376), along with LOS isolated from the B. thetaiotaomicron ΔCPS strain. The ΔCPS strain has wild-type LOS biosynthetic genes, but its lack of CPS yielded mass spectra with a cleaner background. The transposon mutants were created in the background of wild-type B. thetaiotaomicron, and so CPS is present in those preparations. In LOS from the ΔCPS strain, we observed a cluster of peaks around 5,209 m/z, the largest mass detected for any of the samples (Fig. 6). Although instrument-specific limitations prevented us from obtaining a resolution as high as others have observed, the limited degree of resolution that we achieved was sufficient for confirming the approximate masses of LOS molecules in the sample and comparing them to those of the truncated mutants (8,48).
In addition to the peak that we propose corresponds to full-length LOS at 5,209 m/z, the ΔCPS sample has additional peaks at 3,284 and 3,017 m/z, which are likely intermediate species created by partial completion of the biosynthetic pathway. The most truncated transposon mutant in our set, tn3365, has a single predominant peak at 2,961 m/z. LOS from tn3368 does not have this 2,961 m/z peak but instead has peaks at 3,284 m/z (as in the ΔCPS sample) and 3,608 m/z. Interestingly, 2,961, 3,284, and 3,608 m/z are separated from one another by~324 Daltons. The expected mass of a hexose is~162 Daltons, and so we predict that tn3368 makes two LOS molecules that are two and four hexose units longer than the molecule made by tn3365. Finally, LOS from the least truncated transposon mutant that we assayed, tn3376, has its largest peaks around 4,497 and 4,295 m/z, as well as the 3,284 m/z peak that is common to both the tn3368 and the ΔCPS samples. The mass differences between tn3368 and tn3376, as well as between tn3376 and ΔCPS, do not suggest a structural difference as straightforward as the addition of hexoses between tn3365 and tn3368. Given the presence of genes encoding tailoring enzymes in the LOS biosynthetic cluster, we expect that the longer LOS species have modifications like phosphorylation, acetylation, or carbamoylation. All of the transposon mutants have additional peaks between 1,500 and 3,000 m/z that presumably derive from CPS, since these peaks are absent in the ΔCPS sample but present in LOS isolated from the B. thetaiotaomicron ⌬tdk mutant (Fig. S3). Additionally, every sample has a cluster of peaks around 1,688 m/z representing lipid A, which likely derives from in-source fragmentation (48).
With the goal of confirming these results using clean deletion mutants rather than the transposon mutants, we deleted as much of the cluster as we could-BT3363 and BT3365 to BT3380 (we could not obtain mutants of BT3362 and BT3364, consistent with the lack of transposon insertions in these genes) (43). Surprisingly, when we compared LOSs purified from this strain and from the tn3365 mutant on an SDS-PAGE gel, the banding pattern of B. thetaiotaomicron ΔBT3363 ΔBT3365-BT3380 LOS appeared to resemble that of wild-type LOS. However, when we subjected the B. thetaiotaomicron ΔBT3365 ΔBT3365-BT3380 LOS to MALDI-TOF analysis, the mass of the intact glycolipid was around 4,870 Daltons, smaller than the 5,209 Daltons seen in wild-type LOS (Fig. S4). These data suggest that there might be a compensatory mechanism in which  . thetaiotaomicron tnBT3365, B. thetaiotaomicron tnBT3368, and B. thetaiotaomicron tnBT3376 strains were isolated using the large-scale LPS/LOS extraction method. The resulting material was desalted and spotted on a THAP-nitrocellulose matrix for analysis on a Shimadzu Axima Performance MALDI-TOF mass spectrometer in linear negative-ion mode. LOS peaks of interest have been colored, with each color representing a different LOS species present in the different strains. Peaks colored gray are likely derived from capsular polysaccharide (those that do not appear in the B. thetaiotaomicron ΔCPS spectrum) or lipid A in the case of the peak clusters around 1,650 to 1,700 m/z. Bacteroides Lipooligosaccharide Biosynthesis ® the bacterium is able to glycosylate truncated forms of the LOS molecule. Peterson et al. similarly predicted that B. thetaiotaomicron may have the ability to produce an O antigen when this gene cluster is disrupted because the colony morphology of their transposon mutants did not differ from that of wild-type B. thetaiotaomicron (43). However, by our intact LOS MALDI-TOF analysis, we see evidence for new oligosaccharide production only when the cluster is cleanly deleted, rather than when single genes are disrupted by transposon insertion. Our result highlights the limitations of SDS-PAGE analysis in determining structural differences between LOS samples (Fig. S5). Further studies need to be conducted to understand whether B. thetaiotaomicron has an alternative lipid A glycosylation pathway that is unmasked in the absence of most of the LOS oligosaccharide gene cluster.
Predicting other Bacteroides LOS oligosaccharide gene clusters. Having identified the probable B. thetaiotaomicron LOS biosynthetic gene cluster, we hypothesized that it could be used to identify candidate LOS and LPS gene clusters in other Bacteroides species. We used the two essential genes in the cluster, BT3362 (a putative heptosyltransferase) and BT3364 (a putative LPS kinase), as queries in BLAST searches against other Bacteroides genomes and were able to identify similar clusters in many Bacteroides species (Fig. 7). Given that a homologous cluster is found in B. vulgatus, which elaborates a laddered LPS, we expect that B. vulgatus harbors an additional cluster encoding the biosynthesis of the O antigen repeating unit. This would likely be attached to the product of the B. thetaiotaomicron-like core oligosaccharide.
Our results are a first step in characterizing what we expect will be a large amount of biosynthetic and structural heterogeneity among Bacteroides LPS or LOS molecules. Given our understanding of the privileged role that glycolipids play in communicating with the mammalian immune system and the sheer quantity of LPS in the gut, Bacteroides LPS/LOS molecules are likely to be critical mediators in the interaction between commensal microbes and the host. By understanding how these molecules are made, we gain the possibility of manipulating their structure and by extension the host's immune response.

MATERIALS AND METHODS
[M-H] Ϫ ion masses of 1,044.5267, 1,756.9175, 3,492.6357, and 5,728.5931 m/z, respectively (51). The standards were dissolved together in a solution of 0.1% trifluoroacetic acid in water. Because the standard mixture was not in the same solvent as the CMBT matrix mentioned above, 1 l of CMBT matrix was spotted on the target and allowed to dry before 1 l of the standard mixture was spotted on top of the matrix. MS data were collected between 400 and 5,000 m/z, and the resulting spectra were smoothed and baseline corrected using MassLynx software.
Large-scale LPS/LOS extraction. LPS or LOS was isolated from whole bacteria using the hot phenol-water method (52). Briefly, bacteria were grown overnight in a 10-ml culture and then expanded to 2 liters. Cells were harvested once cultures reached an optical density (OD) of at least 0.7 and pelleted by centrifugation at 6,000 ϫ g for 30 min at 4°C. The entire wet cell pellet from the 2-liter culture was suspended in 20 ml water. Separately, the cell suspension and 20 ml of 90% phenol solution in water were each brought up to 68°C with stirring. Once at temperature, the phenol solution was slowly added to the cell suspension. The mixture was stirred vigorously for 30 min at 68°C and then cooled rapidly in an ice water bath for 10 min. The sample was centrifuged at 15,000 ϫ g for 45 min, and the upper aqueous layer was transferred into 1,000-molecular-weight-cutoff (MWCO) dialysis tubing. The sample was dialyzed against 4 liters of water for 4 days, changing the water twice per day. LPS/LOS was pelleted out of the dialysate by ultracentrifugation at 105,000 ϫ g for 4 h. The pellet was resuspended in water and treated with RNase A (Thermo Fisher), DNase I (New England Biolabs), and proteinase K (Thermo Fisher) before repeating the ultracentrifugation step. The pellet was resuspended in water, lyophilized, and stored at Ϫ20°C. LPS/LOS samples that were prepared by this method include the B. thetaiotaomicron ⌬tdk strain in Fig. 4, all samples in Fig. 6, and all samples in Fig. S3, S4, and S5.
Microscale LPS/LOS extraction. The microscale LPS/LOS extraction was used when a large number of samples was needed for SDS-PAGE analysis. In this method, adapted from the work of Marolda and coworkers, bacteria were grown to mid-log phase in 5 ml of medium and pelleted (53). Cell pellets were resuspended in 150 l lysis buffer (0.5 M Tris-hydrochloride, pH 6.8, 2% SDS, 4% ␤-mercaptoethanol) and boiled at 100°C for 10 min. Proteinase K was added to each sample before incubation at 60°C for 1 h. The sample temperature was raised to 70°C, and 150 l prewarmed 90% phenol in water was added. Samples were vortexed three times at 5-min intervals during a 15-min incubation. The samples were immediately cooled on ice for 10 min and centrifuged at 10,000 ϫ g for 1 min. The aqueous layer (~100 l) was pipetted into a clean tube, and 5 volumes of ethyl ether saturated with 10 mM Tris-hydrochloride (pH 8.0) and 1 mM EDTA was added. The samples were vortexed and centrifuged, and the aqueous layer was removed to a clean tube. An appropriate amount of 3ϫ loading dye (0.187 M Tris-hydrochloride, pH 6.8, 6% SDS, 30% glycerol, 0.03% bromophenol blue, 15% ␤-mercaptoethanol) was added, and the samples were stored at Ϫ20°C. LPS/LOS samples that were prepared by this method include B. vulgatus in Fig. 4, all samples in Fig. 5, and all samples in Fig. S2.

SDS-PAGE analysis of LPS.
To visualize LPS/LOS on an SDS-PAGE gel, we used Novex 16% Tricine protein gels (1.0 mm, 12 wells) and 10ϫ Novex Tricine SDS running buffer (Thermo Fisher) (12). For samples prepared by the LPS/LOS microscale extraction, 15 l of the resulting aqueous layer mixed with 3ϫ loading dye was added to each lane. For samples prepared by LPS/LOS large-scale extraction or purchased from InvivoGen, 2.5 g of material was resuspended in 15 l 1ϫ loading dye and the whole volume was added to a lane. Gels were run at 125 V for 90 min at room temperature, stained with Pro-Q Emerald 300 lipopolysaccharide gel stain (Thermo Fisher) per the manufacturer's instructions, and imaged on a Bio-Rad Gel Doc EZ Imager using the SYBR green filter.

MALDI-TOF mass spectrometry analysis of intact LOS. (i) Sample and matrix preparation.
To detect intact LPS by MALDI-TOF MS, we closely followed the technique developed by Phillips et al. adapted to study Neisseria lipooligosaccharides (48). One milligram of lyophilized LOS was dissolved in 100 l 1:3 methanol-water with 5 mM EDTA. Cation exchange beads (Dowex 50WX8, 200 to 400 mesh) were converted to the ammonium form and deposited into 1.5-ml tubes before desalting the LOS. Each sample suspension was added to the beads, vortexed, and centrifuged briefly to pellet the beads. The sample was removed to a clean tube and mixed 9:1 with 100 mM dibasic ammonium citrate before spotting on the target. The matrix was made by mixing a 15-mg/ml solution of nitrocellulose membrane in 1:1 isopropanol-acetone with a 200-mg/ml solution of 2=,4=,6=-trihydroxyacetophenone (THAP) in methanol in a 1:3 ratio. The matrix was deposited by pipetting 1 l within an inscribed circle on the target (Shimadzu) and allowed to dry. Once the matrix had dried completely, 1 l of the sample preparation was added on top of the matrix and allowed to dry.
(ii) Negative-ion MALDI-TOF MS. MALDI-TOF MS analysis was performed on a Shimadzu Axima Performance mass spectrometer with an N 2 laser in linear negative-ion mode. It was calibrated using the same solution of four standards that was used to calibrate the Waters Synapt G2 for lipid A analysisangiotensin II, renin substrate, insulin chain B, and bovine insulin in 0.1% trifluoroacetic acid-and the standards were spotted as described above except on the THAP-nitrocellulose matrix. MS data were collected between 700 and 7,000 m/z, and the resulting spectra were smoothed and baseline corrected using Shimadzu Biotech Launchpad software.

ACKNOWLEDGMENTS
We are deeply indebted to Colleen O'Loughlin and members of the Fischbach group for helpful comments on the manuscript, Daniel Peterson for providing the B. thetaiotaomicron transposon mutants, and Nancy Phillips and Constance John for their guidance on mass spectrometry analysis of lipid A and LOS. Intact LOS mass spectrometry analysis was completed using the Shimadzu Axima Performance MALDI-TOF instrument in the laboratory of William DeGrado at the University of California, San Francisco. Lipid A mass spectrometry analysis on the Waters Synapt G2 HDMS 32k instrument was performed at the University of California, San Francisco Sandler-Moore Mass Spectrometry Core Facility.